CN108707184B - 一种用于凝血酶活性检测的质谱探针及其制备方法和应用 - Google Patents
一种用于凝血酶活性检测的质谱探针及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种用于凝血酶活性检测的质谱探针及其制备方法和应用,属于药物筛选和评价领域。所述的质谱探针,包括氨基酸序列为Phe‑Pro‑Arg‑β‑Ala的多肽及修饰在所述多肽的β‑丙氨酸上的哌嗪类化合物。本发明提供的质谱探针由可被凝血酶特异性识别的多肽Phe‑Pro‑Arg‑β‑Ala与高质谱响应的小分子哌嗪类化合物连接而成,不仅能被凝血酶特异性识别并酶切,同时具有极高的质谱响应,质谱检测准确度高,能够准确反应凝血酶的活性或凝血酶抑制剂的抑制活性,也非常适用于从中药等复杂体系中筛选具有凝血酶抑制活性的化合物。
Description
技术领域
本发明涉及药物筛选和评价领域,具体涉及一种用于凝血酶活性检测的质谱探针及其制备方法和应用。
背景技术
凝血酶(thrombin)属于丝氨酸蛋白酶家族,在机体凝血系统中扮演着重要角色,参与凝血过程中的多个关键环节。凝血酶可促使血浆中的可溶性纤维蛋白原转变为不溶的纤维蛋白,同时激活多个凝血因子,形成血栓。血栓与许多心血管疾病密切相关,如动脉粥样硬化、心肌梗死和冠心病等。因此,凝血酶是治疗心血管疾病的重要靶点之一,直接凝血酶抑制剂(direct thrombin inhibitor)由于无需辅助因子即可抑制凝血酶,受到了药物研发人员的极大重视。
目前使用较为广泛的直接凝血酶抑制剂包括以水蛭素为代表的二价抑制剂(主要为多肽类药物),以及以阿加曲班为代表的一价抑制剂(主要为小分子药物)。此外,从天然产物和传统中药中发现具有凝血酶抑制活性的化合物也是近年来药物筛选领域的一个研究热点。
凝血酶抑制剂的筛选离不开相应的酶活性检测方法。目前,凝血酶活性的检测方法主要包括凝血酶滴定法、抗凝法和发色底物法。
凝血酶滴定法的原理为水蛭素可与凝血酶等比结合形成不可逆的复合物,因此在凝固终点通过水蛭素用量即可计算凝血酶活性。该方法经济简单,易于使用,但重复性和准确度较低且易受干扰,很难用于复杂体系中的药物筛选。
抗凝法通过血凝仪测定凝血酶原时间、凝血酶时间和促凝血酶原激酶时间等,需要实验动物,流程较繁琐。
发色底物法采用光学检测方法测定凝血酶/底物反应前后的吸光度,专属性较高,适用于体内和体外检测,但是,发色底物法通常采用分光光度法测定,灵敏度较低,且易受本底的干扰,难以测定在底物吸收波段有较强吸收的样品,极大地限制了该方法在天然产物筛选中的应用。
在蛋白质组学研究中,为了提高蛋白质谱离子化的效率,常使用化学衍生试剂在肽段上修饰特殊的化学基团。如通过在肽段的氨基、羧基或巯基上引入易于离子化的小分子标签,肽段化学衍生技术可以显著提高肽段的灵敏度和专属性,同时拓展质谱的检测范围。该技术为设计高质谱灵敏度的酶底物提供了一种新思路。
发明内容
本发明的目的在于提供一种的高质谱响应的质谱探针,作为酶底物,被凝血酶特异性识别,通过质谱测定酶切反应前后的探针或酶切产物的量来检测凝血酶的活性。
为实现上述目的,本发明采用如下技术方案:
一种用于凝血酶活性检测的质谱探针,包括氨基酸序列为Phe-Pro-Arg-β-Ala的多肽及修饰在所述多肽的β-丙氨酸上的哌嗪类化合物。
本发明的质谱探针由可被凝血酶特异性识别的多肽与高质谱响应的小分子连接而成。所述多肽的氨基酸序列为:苯丙氨酸-脯氨酸-精氨酸-β-丙氨酸(SEQ ID NO.1),所述的高质谱响应的小分子为哌嗪类化合物,哌嗪类化合物的-NH与多肽链上的β-丙氨酸的羧基缩合。
凝血酶酶切位点为精氨酸与β-丙氨酸之间的酰胺键,因此,本发明质谱探针的酶切产物为苯丙氨酸-脯氨酸-精氨酸和β-丙氨酸-哌嗪类化合物。由于哌嗪类化合物具有很强的质谱响应,因此通过测定反应前后该探针或酶切产物β-丙氨酸-哌嗪类化合物的量,即可检测凝血酶的活性。
作为优选,所述哌嗪类化合物为1-(2-嘧啶基)哌嗪、1-(4-吡啶基)哌嗪或1-(1-甲基-4-吡啶基)哌嗪。
更为优选,所述哌嗪类化合物为1-(2-嘧啶基)哌嗪。所述质谱探针的分子结构式如式(Ⅰ)所示,
作为优选,所述多肽中的苯丙氨酸为D型。相比L型,D型苯丙氨酸可以提高探针与酶的反应速率。
本发明还提供了一种合成所述质谱探针的制备方法,包括:
(1)采用固相法合成氨基酸序列为Phe-Pro-Arg-β-Ala的多肽;
(2)将所述多肽溶于二氯甲烷,加入二异丙基乙胺调pH值到中性,再加入5倍摩尔量的1-羟基苯并三唑和哌嗪类化合物,充分溶解,然后加入5倍摩尔量的N,N-二异丙基碳二亚胺进行反应,反应结束后旋蒸二氯甲烷,纯化制得所述的质谱探针。
步骤(1)中,以2-Chlorotrityl Chloride Resin树脂为载体,Fmoc保护的氨基酸为原料,O-苯并三氮唑-四甲基脲六氟磷酸盐(HBTU)为缩合剂,合成氨基酸序列为Phe-Pro-Arg-β-Ala的多肽。
步骤(2)中,液相反应将哌嗪类化合物连接到所述多肽链上,所述反应时间为3h,反应结束后,在50℃以下旋蒸二氯甲烷,切割得到质谱探针粗品,再利用高效液相色谱法(HPLC)纯化,制得所述质谱探针。
本发明的另一个目的是提供所述质谱探针的应用。所述质谱探针作为酶解底物,被凝血酶特异性识别,酶切成苯丙氨酸-脯氨酸-精氨酸和β-丙氨酸-哌嗪类化合物。通过质谱仪或液质联用仪测定酶切反应前后质谱探针或酶切产物β-丙氨酸-哌嗪类化合物的量,利用差值表征凝血酶的活性。
因此,本发明提供了所述的质谱探针在制备检测凝血酶活性的试剂盒中的应用。
本发明还提供了所述的质谱探针在制备筛选凝血酶抑制剂的试剂盒中的应用。
本发明提供的质谱探针具有极高的质谱响应,极大提高了检测的灵敏度,另外,由于质谱探针和β-丙氨酸-哌嗪类化合物的分子量是确定的,该检测方法的选择性和专属性很高,非常适用于从中药等复杂体系中筛选具有凝血酶抑制活性的化合物。
本发明还提供了所述的质谱探针在评价药物的凝血酶抑制活性中的应用。利用本发明的质谱探针检测不同批次同种药物的凝血酶抑制活性,进而评价不同批次药物的质量。
本发明具备的有益效果:
本发明提供的质谱探针由可被凝血酶特异性识别的多肽Phe-Pro-Arg-β-Ala与高质谱响应的小分子哌嗪类化合物连接而成,不仅能被凝血酶特异性识别并酶切,同时具有极高的质谱响应,质谱检测准确度高,能够准确反应凝血酶的活性或凝血酶抑制剂的抑制活性,也非常适用于从中药等复杂体系中筛选具有凝血酶抑制活性的化合物。
附图说明
图1为凝血酶质谱探针纯度分析的HPLC色谱图。
图2为凝血酶质谱探针结构确证的HPLC-IT-MS基峰离子流图,其中右上角附图为质谱结果图。
图3为凝血酶质谱探针测定不同浓度凝血酶的活性。
图4为用凝血酶质谱探针测定凝血酶阳性药AEBSF-HCl的抑制率。
具体实施方式
下面通过具体实施案例对本发明做进一步描述。
实施例1
凝血酶质谱探针的合成
凝血酶质谱探针的结构为D-苯丙氨酸—脯氨酸—精氨酸—β-丙氨酸—1-(2-嘧啶基)哌嗪(D-Phe-Pro-Arg-β-Ala-PP),通过固相合成法合成肽段,用液相反应连接上1-(2-嘧啶基)哌嗪(PP)基团,主要包括以下步骤:
一.树脂溶胀
称取取代度为0.4mmol/g的2-Chlorotrityl Chloride Resin树脂,将树脂放入反应管中,加二氯甲烷,振荡30min。
二.接第一个氨基酸
抽滤掉溶剂,加入3倍摩尔过量的Fmoc-β-Ala-OH,再加入5倍摩尔过量的二异丙基乙胺,最后加入少量二甲基甲酰胺溶解,振荡1h。用二甲基甲酰胺和二氯甲烷交替清洗6遍。
三.脱保护
加入20%哌啶二甲基甲酰胺溶液,反应5min,去掉溶剂,再次加入20%哌啶二甲基甲酰胺溶液,反应15min。反应后用二甲基甲酰胺、甲醇和二甲基甲酰胺清洗两次。
四.缩合
3倍摩尔过量的Fmoc-L-Arg(Pbf)-OH和3倍摩尔过量的苯并三氮唑-四甲基脲六氟磷酸盐(HBTU),用少量二甲基甲酰胺溶解,加入反应管,立即加入5倍摩尔过量二异丙基乙胺,反应60min。反应后用二甲基甲酰胺、甲醇和二甲基甲酰胺清洗两次。重复以上步骤,从右到左依次连接上Pro和Phe,连接完成后用甲醇洗4次,抽干10min。
五.切割、吹干、洗涤
配制切割液(TFA:水:DCM:TIS=95:2.5:2:0.5),从树脂上切割多肽,溶液用氮气吹干,用乙醚将多肽析出,再用乙醚洗涤6次,常温挥干,得到全保护粗品多肽。
六.液相反应接PP
将全保护粗品多肽用二氯甲烷溶解,加入二异丙基乙胺调pH值到中性,加入五倍摩尔过量1-羟基苯并三唑(HOBT)和PP充分溶解,然后加五倍摩尔过量N,N-二异丙基碳二亚胺,反应3h。温度控制在50℃以下旋蒸二氯甲烷,加入切割液切割多肽,得粗品探针。
七.HPLC纯化多肽
用HPLC纯化粗品探针,将纯化后的溶液冻干,-20℃保存待用。
实施例2
凝血酶质谱探针的化学表征
采用实施例1所述的方法合成凝血酶质谱探针,用HPLC对探针的纯度进行分析。分析条件为:捷伦1200HPLC色谱系统,配备可变波长检测器(VWD),检测波长214nm;色谱柱,Kromasil C18(4.6mm×150mm,5μm);流动相为0.1%三氟乙酸-水(A)和0.1%三氟乙酸-乙腈(B);流速1.0mL/min;等度洗脱,0-25min,5-70%B;进样量,10μL。
将凝血酶质谱探针用纯水溶解后,10000rpm离心5分钟,进样。HPLC色谱图如图1所示。从图上可以看出,合成的凝血酶质谱探针纯度很高,相对峰面积占比>95%,可以用于后续的实验和研究。
此外,我们进一步用HPLC-MS对探针的结构进行了确证。分析条件为:安捷伦1100HPLC色谱系统,串联LCQ Deca XPplus离子阱质谱(HPLC-IT/MS);ESI离子源;正离子模式;质荷比(m/z),100-1000;毛细管电压,15V;源电压,3kV;毛细管温度350℃;鞘气(N2)60arb;辅助气(N2)20arb;色谱柱,Zorbax SB C18(4.6mm×100mm,1.8μm);流动相为0.05%甲酸-水(A)和0.05%甲酸-乙腈(B);流速0.4mL/min;洗脱梯度为0-5min,1%B;5-40min,1-30%B;40-45min,30-100%B;45-50min,100%B;进样量,5μL。
凝血酶质谱探针的HPLC-IT/MS色谱图如图2所示。从质谱图上可以看到准分子离子峰[M+H]+(m/z 636.3)以及双电荷离子[M+2H]2+(m/z318.9)。这些离子与探针的结构相吻合,也进一步确证了探针的结构。
实施例3
凝血酶质谱探针在凝血酶活性检测中的应用
采用pH=8.3,10mM的Tris-HCl作为缓冲液。取凝血酶质谱探针和不同浓度的凝血酶溶液各20μL,缓冲液160μL,置于1.5mL的离心管中,使凝血酶质谱探针的终浓度为0.02mM,凝血酶的终浓度为0.01-0.2U/mL,在37℃孵育2h。反应结束后加入400μL甲醇终止反应。溶液涡旋、离心,用实施例2所述HPLC-IT/MS方法分析,以反应前后凝血酶质谱探针的峰面积差值反映凝血酶活性,结果如图3所示。从图上可以看出,在该浓度范围内,凝血酶的浓度(活性)和反应前后凝血酶质谱探针峰面积的差值有良好的线性关系,即凝血酶浓度越高,被酶切的探针越多,峰面积的差值也越高。因此,该探针可以用于凝血酶的活性检测。
实施例4
凝血酶质谱探针在凝血酶抑制剂筛选中的应用
采用pH=8.3,10mM的Tris-HCl作为缓冲液。取凝血酶质谱探针和凝血酶溶液各20μL,不同浓度的AEBSF-HCl(凝血酶抑制剂)50μL,缓冲液110μL,置于1.5mL的离心管中,使凝血酶质谱探针的终浓度为0.02mM,凝血酶的终浓度为0.2U/mL,AEBSF-HCl的终浓度为0.05-263μM,在37℃孵育2h。反应结束后加入400μL甲醇终止反应。溶液涡旋、离心,用实施例2所述HPLC-IT/MS方法分析,用以下公式计算酶抑制率:
凝血酶抑制率(%)=[1-(ΔPinhibitor/ΔPblank)]×100,
其中ΔPinhibitor和ΔPblank分别为抑制剂组和对照组(不加抑制剂)反应前后凝血酶质谱探针的峰面积。
测试结果如图4所示。从图上可以看出,随着AEBSF-HCl浓度的上升,它对凝血酶的抑制率也逐渐增强。这表明凝血酶探针可以较好地反映抑制剂对凝血酶的抑制作用,可以用于凝血酶抑制剂的筛选。
实施例5
凝血酶质谱探针在具有凝血酶抑制作用的中药质量评价中的应用
取5个不同批次的丹参药材粉末各0.5g,精密称定,加甲醇-水(体积比为8:2)混合溶液50mL,超声提取30min,用离心浓缩机干燥,获得丹参提取物粉末,实验前用Tris-HCl缓冲液配制成样品溶液。
采用pH=8.3,10mM的Tris-HCl作为缓冲液。取凝血酶质谱探针和凝血酶溶液各20μL,丹参样品溶液50μL,缓冲液110μL,置于1.5mL的离心管中,使凝血酶质谱探针的终浓度为0.02mM,凝血酶的终浓度为0.2U/mL,丹参的终浓度为2mg/mL,在37℃孵育2h。反应结束后加入400μL甲醇终止反应。溶液涡旋、离心,用实施例2所述HPLC-IT/MS方法分析,用以下公式计算酶抑制率:
凝血酶抑制率(%)=[1-(ΔPinhibitor/ΔPblank)]×100,
其中ΔPinhibitor和ΔPblank分别为丹参样品组和对照组(不加抑制剂)反应前后凝血酶质谱探针的峰面积。
结果表明,5个批次丹参凝血酶的抑制率分别为41%、48%、54%、51%、45%。这表明丹参对凝血酶有较强的抑制作用,而凝血酶也与丹参活血化瘀的功效相匹配。因此,采用该方法可以较好地反映丹参的生物活性,也可用于丹参的质量评价。
以上所述仅为本发明专利的具体实施案例,但本发明专利的技术特征并不局限于此,任何相关领域的技术人员在本发明的领域内,所作的变化或修饰皆涵盖在本发明的专利范围之中。
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<110> 浙江大学
<120> 一种用于凝血酶活性检测的质谱探针及其制备方法和应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 4
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Phe Pro Arg Ala
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Claims (6)
1.一种用于凝血酶活性检测的质谱探针,其特征在于,由氨基酸序列为Phe-Pro-Arg-β-Ala的多肽及修饰在所述多肽的β-丙氨酸上的哌嗪类化合物组成;所述哌嗪类化合物为1-(2-嘧啶基)哌嗪、1-(4-吡啶基)哌嗪或1-(1-甲基-4-吡啶基)哌嗪,哌嗪类化合物的-NH与多肽链上的β-丙氨酸的羧基缩合;所述多肽中的苯丙氨酸为D型。
3.如权利要求1-2任一项所述的质谱探针的制备方法,其特征在于,包括:
(1)采用固相法合成氨基酸序列为Phe-Pro-Arg-β-Ala的多肽;
(2)将所述多肽溶于二氯甲烷,加入二异丙基乙胺调pH值到中性,再加入5倍摩尔量的1-羟基苯并三唑和哌嗪类化合物,充分溶解,然后加入5倍摩尔量的N,N-二异丙基碳二亚胺进行反应,反应结束后旋蒸二氯甲烷,纯化制得所述的质谱探针。
4.如权利要求1-2任一项所述的质谱探针在制备检测凝血酶活性的试剂盒中的应用。
5.如权利要求1-2任一项所述的质谱探针在制备筛选凝血酶抑制剂的试剂盒中的应用。
6.如权利要求1-2任一项所述的质谱探针在体外评价药物的凝血酶抑制活性中的应用。
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